In animals and the more advanced microorganisms, the enzymes involved in de novo synthesis of fatty acids are integrated into a multienzyme complex. We have found that the component activities of mammalian fatty acid synthetases are linked together covalently on two polyfunctional polypeptides. Using limited proteolysis we detached the two thioesterase I domains, one from each subunit, in active form. The thioesterase I domains occupy terminal positions on the two polypeptide chains; by identifying the terminal amino acid residues on the native multienzyme and its proteolytic products, we will determine whether the theoesterase I domain is at the C or N terminus. The thioesterase I component of the multienzyme is responsible for termination, at a particular chain length, of growth of the acyl moiety on the multienzyme. From a study of its properties, we hope to establish how the product specificity of the multienzyme complex is regulated. Whereas the isolated fatty acid synthetases from all animal tissue synthesize mainly palmitic acid, in lactating mammary gland the product specificity is shifted toward shorter chain length fatty acids by the presence of a second enzyme, thioesterase II which is not part of the multienzyme. Thioesterase II is present uniquely in mammary gland and is not found in tissues which make only long chain fatty acids. We will compare the structure, specificity and amino acid sequence of thioesterases I and II to establish a basis for their different functions and to gain an insight into their evoluationary origins. We propose to study the mechanism of interaction between fatty acid synthetase and thioesterase II, to determine how the product specificity of the multienzyme is modified by the second enzyme.